Project Summary ? Project 1 T cell responses to viruses are initiated, function and maintained in diverse tissues sites; however, our knowledge of human anti-viral T cells is largely derived from the sampling of peripheral blood, containing only 2-3% of the total T cell complement. Studies in mouse models have revealed the importance of tissue- localized T cell responses in viral clearance and long-term maintenance of protective immunity in the form of a subset of non-circulating, tissue-resident memory T (TRM) cells. The discovery of TRM raises the critical question of how the circulating T cell response is related to that in tissues. My laboratory has set up a novel tissue resource and protocol with the organ procurement organization for New York City to obtain multiple lymphoid and mucosal tissues from individual organ donors, providing an unprecedented opportunity to study immune cells and responses in tissues and circulation. In five years of this protocol, we have optimized the acquisition and isolation of cells from tissues of 250 donors representing all ages of life, and have discovered novel aspects of how human T cells differentiate, become compartmentalized and function in tissues and circulation at different life stages. These tissues also provide a new opportunity to study ongoing anti-viral T cell responses in situ, as their serological profile for the prevalent persisting herpesviruses is known, including human CMV (hCMV; 60% donors) which infects multiple tissues and establishes lifelong latency. Human CMV requires active T cell responses to be controlled?a significant frequency of blood CD8 T cells (5- 30%) are CMV-specific in seropositive individuals, and immunosuppression due to transplantation, cancer or HIV infection can trigger CMV reactivation leading to virus dissemination, end organ failure and death. We propose a novel investigation of CMV-specific T cell responses in circulation and diverse tissues of individual donors to understand on a fundamental level how tissue location impacts anti-viral T cell responses to CMV, and as a ?model system? for assessing tissue-mediated control of anti-viral immunity. Our central hypothesis is that the functionality of T cells specific for CMV in healthy humans will vary depending on tissue site which will be further impacted by virus persistence and age. In the proposed project, we will profile CMV-specific T cell responses from diverse human lymphoid, mucosal and endocrine tissues sites obtained through the Clinical Core, together with blood samples from transplant patients who developed CMV viremia for understand how tissue impacts the T cell response, define tissue-specific signatures for T cell responses and identify whether peripheral blood T cell responses during active infection will contain traces of their tissue of origin. We will profile using whole transcriptome profiling on the population and single cell level by Core A, high dimensional analysis of protein expression by CyTOF also through Core A, with all data curated and analyzed through the data management and analysis (DMAC) core. Results from this study will elucidate tissue signatures of T cell- mediated responses and a new understanding of how circulating responses represent those in tissues.